Antibacterial composition and application thereof

Abstract

The invention relates to an antibacterial composition and application thereof, and belongs to the technical field of biological medicine. The invention provides an antibacterial composition, which is prepared from amino acid and esculentin, and the mass ratio of the amino acid to the esculentin is (5-15): 1. According to the invention, the amino acid and the eriocycline are combined into the antibacterial composition, and in-vitro and in-vivo experiments prove that the antibacterial composition has more excellent effects of inhibiting and killing drug-resistant bacteria compared with a single drug of the amino acid or the eriocycline, and can be applied to products for resisting the drug-resistant bacteria. Meanwhile, animal experiments prove that the antibacterial composition can improve the expression level of proinflammatory factors (TNF-alpha, CXCL12 and the like) caused by drug-resistant bacteria, has a certain anti-inflammatory effect, and brings new inspiration to the field of treatment of drug-resistant bacteria infection.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and particularly relates to an antibacterial composition and its application. Background Art

[0002] In recent years, the rapidly increasing infections caused by drug-resistant bacteria have seriously threatened human life and health. As the world's first fluorocycline antibacterial drug, eravacycline has a fluorine atom introduced into the core D ring of tetracycline, and its drug performance has been improved to a certain extent. Due to its strong antibacterial activity, broad antibacterial spectrum, high tissue concentration and other advantages, it has been recommended by many authoritative domestic and foreign guidelines for the empirical treatment of multi-drug resistant bacteria (MDR). For example, the "Guidelines for the Treatment of MDR Gram-negative Bacilli" published by ESCMID and IDSA in 2022 recommend eravacycline for empirical treatment, which can become the cornerstone treatment plan for MDR Gram-negative bacterial infections including carbapenem-resistant ones.

[0003] The existing strategies for controlling drug-resistant bacterial infections mainly focus on the development of new antibacterial drugs. However, the development cycle of new antibacterial drugs is long, and drug-resistant bacteria, even super drug-resistant bacteria, are selected during the use process, resulting in the situation that clinically infected patients have "no available drugs" from time to time. Even though eravacycline was launched in China in 2023, with the widespread use of the drug, drug-resistant bacteria have gradually been discovered.

[0004] Currently, antibacterial drugs are often used in combination with enzyme inhibitors to control drug-resistant bacteria. For example, β-lactamase inhibitors are widely used in clinical practice in combination with β-lactam antibacterial drugs to promote the bactericidal efficiency of the latter. However, since β-lactamase inhibitors also have bacteriostatic effects, bacteria have developed resistance to them. Therefore, there is an urgent need to develop green and effective strategies for controlling drug-resistant bacteria. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a green and effective antibacterial composition for controlling drug-resistant bacteria and its application.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] In the first aspect, the present invention provides an antibacterial composition, comprising an amino acid and eravacycline, and the mass ratio of the amino acid to eravacycline is amino acid:eravacycline = (5 - 15):1.

[0008] The present invention combines an amino acid and eravacycline into an antibacterial composition. Through in vitro and in vivo experiments, it is confirmed that compared with the amino acid or eravacycline alone, the antibacterial composition has a more excellent effect of inhibiting and killing drug-resistant bacteria and can be applied in products against drug-resistant bacteria. At the same time, the present invention also confirms through animal experiments that the antibacterial composition can improve the expression levels of pro-inflammatory factors (such as TNF-α, CXCL12, etc.) caused by drug-resistant bacteria, and has a certain anti-inflammatory effect, bringing new inspiration to the field of treating drug-resistant bacterial infections.

[0009] As a preferred embodiment of the antibacterial composition of the present invention, the mass ratio of the amino acid to eravacycline is amino acid: eravacycline = 10:1.

[0010] As a preferred embodiment of the antibacterial composition of the present invention, the amino acid includes at least one of methionine, phenylalanine, glycine, alanine, and aspartic acid.

[0011] As a preferred embodiment of the antibacterial composition of the present invention, the amino acid is methionine.

[0012] In a second aspect, the present invention provides the use of the above antibacterial composition in the preparation of antibacterial products.

[0013] As a preferred embodiment of the use of the present invention, the antibacterial product can inhibit drug-resistant bacteria.

[0014] As a preferred embodiment of the use of the present invention, the drug-resistant bacteria include at least one of carbapenem-resistant pathogenic bacteria and vancomycin-resistant pathogenic bacteria. According to in vitro experiments, the antibacterial composition of the present invention has a broad-spectrum anti-drug-resistant bacteria effect, proving that the antibacterial product containing the antibacterial composition of the present invention can also broadly inhibit and kill drug-resistant bacteria.

[0015] As a preferred embodiment of the use of the present invention, the carbapenem-resistant pathogenic bacteria include at least one of carbapenem-resistant Klebsiella pneumoniae, carbapenem-resistant Escherichia coli, and carbapenem-resistant Acinetobacter baumannii.

[0016] As a preferred embodiment of the use of the present invention, the vancomycin-resistant pathogenic bacteria include at least one of vancomycin-resistant Staphylococcus epidermidis and vancomycin-resistant Staphylococcus haemolyticus.

[0017] As a preferred embodiment of the use of the present invention, the products include at least one of drugs, foods, health products, and biological products.

[0018] In a third aspect, the present invention provides an antibacterial product, comprising the above antibacterial composition and excipients.

[0019] As a preferred embodiment of the antibacterial product of the present invention, the dosage form of the antibacterial product includes, but is not limited to, at least one of solid dosage forms, liquid dosage forms, semi-solid dosage forms, and other special dosage forms.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] The present invention combines an amino acid and eravacycline into an antibacterial composition. Through in vitro and in vivo experiments, it is confirmed that compared with the amino acid or eravacycline alone, the antibacterial composition has a more excellent effect of inhibiting and killing drug-resistant bacteria and can be applied in products against drug-resistant bacteria. At the same time, the present invention also confirms through animal experiments that the antibacterial composition can improve the expression levels of pro-inflammatory factors (such as TNF-α, CXCL12, etc.) caused by drug-resistant bacteria, and has a certain anti-inflammatory effect, bringing new inspiration to the field of treating drug-resistant bacterial infections. Description of the Drawings

[0022] Figure 1 Effects of the combination of compound amino acids and fat emulsion injection with eravacycline on carbapenem-resistant Klebsiella pneumoniae CRKP8 in Example 1 of the present invention. In each treatment, from left to right are 0, 0.5, 1, 2, 4, 8 μg / mL of eravacycline, and from top to bottom are CRKP8 diluted 10 (i.e., bacterial concentration is 10 -1 ), 100 (i.e., bacterial concentration is 10 -2 ), 1000 (i.e., bacterial concentration is 10 -3 ), and 10,000 times (i.e., bacterial concentration is 10 -4 );

[0023] Figure 2 Effects of the combination of different amino acids and eravacycline on carbapenem-resistant Klebsiella pneumoniae CRKP8 in Example 2 of the present invention. In each treatment, from top to bottom are 10 (i.e., bacterial concentration is 10 -1 ), 100 (i.e., bacterial concentration is 10 -2 ), 1000 (i.e., bacterial concentration is 10 -3 ), and 10,000 times (i.e., bacterial concentration is 10 -4 ) of CRKP8;

[0024] Figure 3 Effects of the combination of methionine and eravacycline on different types of drug-resistant bacteria in Example 3 of the present invention. In each treatment, from top to bottom are drug-resistant bacteria diluted 10, 100, 1000, and 10,000 times;

[0025] Figure 4 Statistical results of the survival rates of mice with abdominal infection models under different treatments in the efficacy example of the present invention;

[0026] Figure 5This is the statistical result of the number of carbapenem-resistant Klebsiella pneumoniae CRKP8 in the viscera of mice with abdominal infection models under different treatments in the efficacy examples of the present invention. From top to bottom in each organ are tissue suspensions diluted 10, 100, 1000, and 10000 times.

[0027] Figure 6 This is the HE staining result of the viscera of mice with abdominal infection models under different treatments in the efficacy examples of the present invention.

[0028] Figure 7 This is the statistical result of the expression levels of pro-inflammatory factors in the viscera of mice with abdominal infection models under different treatments in the efficacy examples of the present invention.

[0029] In the above figures, "ERV" refers to eravacycline, "18AA-Ⅱ" refers to compound amino acids, "C14-24" refers to fat emulsion injection, "AA" refers to amino acids, "CREC9" refers to carbapenem-resistant Escherichia coli CREC9, "CRAB8" refers to carbapenem-resistant Acinetobacter baumannii CRAB8, "VRS5" refers to vancomycin-resistant Staphylococcus xylosus VRS5, "VRS6" refers to vancomycin-resistant Staphylococcus hominis VRS6, and "VRS7" refers to vancomycin-resistant Staphylococcus haemolyticus VRS7.

[0030] Those with the "*" label indicate significant differences between the two groups (p < 0.05). Detailed implementation manners

[0031] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0032] Other materials, reagents, etc. used in the examples, comparative examples, and efficacy examples can be obtained from commercial channels without special instructions.

[0033] The carbapenem-resistant Klebsiella pneumoniae CRKP8, carbapenem-resistant Escherichia coli CREC9, carbapenem-resistant Acinetobacter baumannii CRAB8, vancomycin-resistant Staphylococcus xylosus VRS5, vancomycin-resistant Staphylococcus hominis VRS6, and vancomycin-resistant Staphylococcus haemolyticus VRS7 selected in the following examples were all isolated and purified by the inventor's team.

[0034] Kunming mice were purchased from the Experimental Animal Center of Sun Yat-sen University in Guangzhou.

[0035] Example 1

[0036] To determine the metabolites that can improve the efficiency of eravacycline in inhibiting drug-resistant bacteria, compound amino acids and fat emulsion injection, which are commonly used clinically, were respectively combined with eravacycline, and carbapenem-resistant Klebsiella pneumoniae CRKP8 (hereinafter referred to as CRKP8) was used as the drug-resistant bacteria for in vitro bactericidal experiments. The specific protocol is as follows:

[0037] The activated CRKP8 was prepared into a bacterial suspension with a concentration of 10 7 CFU / mL and divided into three groups: ERV, ERV + 18AA-II, and ERV + C14-24. The final concentration of 18AA-II was 0.85% (v / v), and the final concentration of C14-24 was 2% (v / v); each group contained 6 test tubes, and ERV was added to make its final concentration 0, 0.5, 1, 2, 4, 8 μg / mL; the bactericidal experiment was carried out on a shaker for 6 h. After the time ended, the bacterial suspension was taken for 10-fold serial dilution, and then 5 μL of the diluted bacterial suspension was inoculated onto an LB agar plate and cultured at 37°C for 16 h to observe the growth of CRKP8. The results are shown in Figure 1 .

[0038] As Figure 1 shown, in the ERV alone group, as the antibiotic dose increased, the drug-resistant bacteria were gradually killed. When the concentration reached 1 μg / mL, the survival rate of the strain was about 85%, and when it reached 8 μg / mL, the survival rate dropped to 5%. In the ERV + C14-24 group, as the antibiotic dose increased, the drug-resistant bacteria were gradually killed, but there was no difference compared with ERV alone, indicating that the fat emulsion (C14-24) failed to effectively enhance the antibacterial activity of ERV. In the ERV + 18AA-II group, when ERV was 1 μg / mL, the survival rate of the drug-resistant bacteria had decreased to 40%, and when it was 8 μg / mL, the survival rate of the drug-resistant bacteria had decreased to 0.02%. This shows that compound amino acids (18AA-II) have a significant synergistic effect on ERV, greatly improving the sensitivity of bacteria to eravacycline.

[0039] In summary, compound amino acids have the potential to improve the killing effect of eravacycline on CRKP8.

[0040] Example 2

[0041] To determine which amino acids can enhance the antibacterial effect of eravacycline, methionine, phenylalanine, glycine, alanine, and aspartic acid were screened for combination with eravacycline. An in vitro bactericidal experiment was conducted using carbapenem-resistant Klebsiella pneumoniae CRKP8 (hereinafter referred to as CRKP8) as the drug-resistant bacterium. The experimental protocol was similar to that of Example 1, except that the compound amino acid was replaced with methionine, phenylalanine, glycine, alanine, and aspartic acid. Each group included four tubes: a blank group (PBS), different amino acid groups (AA), an eravacycline group (ERV), and a combined group (AA + ERV). The final concentration of ERV was 2 μg / mL, and the final concentrations of the above-mentioned amino acids were 20 μg / mL respectively. The results are shown in Figure 2 .

[0042] As Figure 2 shown, except for methionine, the other four amino acids did not show obvious synergistic effects; in the presence of 20 μg / mL methionine, the killing effect of eravacycline on CRKP8 strains was significantly increased by about 10 times, indicating that the combination of methionine and eravacycline can synergistically improve the efficacy of killing CRKP strains.

[0043] Example 3

[0044] To verify whether the combination of methionine and eravacycline has a broad-spectrum effect on killing drug-resistant bacteria, methionine and eravacycline were combined according to the method of Example 2, and the effects of this combination on imipenem-resistant Escherichia coli CREC9, carbapenem-resistant Acinetobacter baumannii CRAB8, vancomycin-resistant Staphylococcus xylosus VRS5, vancomycin-resistant Staphylococcus hominis VRS6, and vancomycin-resistant Staphylococcus haemolyticus VRS7 were measured. The results are shown in Figure 3 .

[0045] As Figure 3 shown, the combination of methionine and eravacycline has a synergistic killing effect on CREC9, CRAB8, VRS5, VRS7, and VRS6, increasing the bactericidal efficiency of eravacycline by 4 times (CREC9), 15.6 times (CRAB8), 13 times (VRS5), 30 times (VRS6), and 12 times (VRS7) respectively, indicating that the combination of methionine and eravacycline has a broad-spectrum effect on killing drug-resistant bacteria.

[0046] Example 4

[0047] An embodiment of the antibacterial composition of the present invention. The antibacterial composition in this embodiment includes methionine and eravacycline, and the mass ratio of methionine to eravacycline is methionine:eravacycline = 10:1.

[0048] Example 5

[0049] An embodiment of the antibacterial composition of the present invention comprises methionine and eracycline, and the mass ratio of methionine to eracycline is methionine:eracycline=5:1.

[0050] Example 6

[0051] An embodiment of the antibacterial composition of the present invention comprises methionine and eracycline, and the mass ratio of methionine to eracycline is methionine:eracycline=15:1.

[0052] Effect example

[0053] In order to explore the efficacy of the antibacterial composition obtained in Example 4 in killing drug-resistant bacteria in vivo, this experiment constructed an abdominal infection model for verification. The specific scheme is as follows:

[0054] 1. Select Kunming mice and inject 10 7 CFU of CRKP8 was used to construct an intraperitoneal infection model.

[0055] 2. The intraperitoneal infection model mice obtained in step 1 were divided into 4 treatment groups and injected with PBS, methionine (50 mg / kg), eracycline (5 mg / kg) or the antibacterial composition obtained in Example 4 (methionine 50 mg / kg + eracycline 5 mg / kg). The survival rate of the mice was continuously observed and counted after injection until 36 hours after injection. The mice were killed after blood samples were collected, and the liver, kidney, and spleen tissues were taken out and ground into a suspension. The amount of CRKP8 was determined according to the method of Example 1. At the same time, the liver, kidney, and spleen were stained with HE and the expression levels of pro-inflammatory factors (TNF-α, CXCL12) were detected by immunohistochemistry. The results are shown in Figures 4 - 7 .

[0056] like Figures 4 - 5 As shown, during the 36h observation period, the survival rate of mice in the control group was only 28.6%; while the mortality rate of mice treated with methionine alone did not change significantly compared with the control group. The survival rate of the group treated with elacycline alone increased to 57.1%; particularly notably, the survival rate of mice treated with the antibacterial composition further increased significantly to 85.7%. Further analysis of samples of major organs in the abdominal cavity (including the liver, spleen and kidneys) found that treatment with elacycline alone did not significantly reduce the bacterial load in the organs, and there was no significant difference compared with the untreated group. However, treatment with methionine alone led to an increase in the bacterial load in the organs, especially in the spleen. In contrast, the antibacterial composition significantly improved the efficiency of bacterial clearance, resulting in a significant reduction in the bacterial load in the organs.

[0057] like Figure 6As shown, untreated mice showed obvious symptoms of liver inflammatory infiltration and spleen congestion; while mice treated with the antibacterial composition showed significant symptom relief, further indicating that the antibacterial composition can also achieve the effect of killing drug-resistant bacteria in vivo.

[0058] As Figure 7 shown, it was found that the expression of TNF-α was enhanced in the liver tissue sections of the untreated group, and the expressions of TNF-α and CXCL12 in the spleen were strong. After receiving the combination treatment, the above indicators were significantly improved. These research results indicate that the combined application of methionine and eravacycline can reduce the inflammatory response. Combining with the previous experimental results, it is confirmed that the combination of methionine and eravacycline as a composition shows great potential in effectively combating drug-resistant bacterial infections.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An antibacterial composition, characterized in that The invention comprises amino acid and eracycline, wherein the mass ratio of the amino acid to the eracycline is amino acid:eracycline=(5-15):

1.

2. The antibacterial composition according to claim 1, characterized in that The mass ratio of the amino acid to eracycline is amino acid:eracycline=10:

1.

3. The antibacterial composition according to claim 1, characterized in that The amino acid includes at least one of methionine, phenylalanine, glycine, alanine and aspartic acid.

4. Use of the antibacterial composition according to any one of claims 1 to 3 in the preparation of antibacterial products.

5. The use according to claim 4, characterized in that The antibacterial product can inhibit drug-resistant bacteria.

6. The use according to claim 5, characterized in that The drug-resistant bacteria include at least one of carbapenem-resistant pathogens and vancomycin-resistant pathogens.

7. The use according to claim 6, characterized in that The carbapenem-resistant pathogenic bacteria include at least one of carbapenem-resistant Klebsiella pneumoniae, carbapenem-resistant Escherichia coli and carbapenem-resistant Acinetobacter baumannii.

8. The use according to claim 6, characterized in that The vancomycin-resistant pathogens include at least one of vancomycin-resistant Staphylococcus epidermidis and vancomycin-resistant Staphylococcus hemolyticus.

9. The use according to claim 4, characterized in that The product comprises at least one of medicine, food, health care product and biological product.

10. An antibacterial product, characterized in that: The invention comprises the antibacterial composition and auxiliary materials as described in any one of claims 1 to 3.

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